ANTIBODY COMPOSITIONS TARGETING NON-PHOSPHORYLATED a-SYNUCLEIN AGGREGATES

ABSTRACT

The present specification provides a monoclonal antibody that specifically binds aggregated, non-phosphorylated α-synuclein and a hybridoma producing it. Also disclosed are methods of generating antibodies that specifically binds aggregated, non-phosphorylated α-synuclein and uses thereof. Uses of anti-α-synuclein antibody in detection and diagnostic assays, and for prophylaxis or therapy of α-synuclein-associated neurodegenerative diseases, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application No. 63/079,767, filed Sep. 17, 2020, which is incorporated herein by reference in its entirety.

BACKGROUND

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are collectively known as Lewy body disease and are the second most common neurodegenerative disorder after Alzheimer's disease (AD). The neuropathological hallmark of Lewy body disease is the intracellular aggregation of the protein alpha-synuclein (α-syn) into spherical cytoplasmic inclusions termed Lewy bodies, but aggregates are also observed in neuronal processes as Lewy neurites.

Alpha-synuclein is thought to play a central role in the pathobiology of Lewy body disease. Single point mutations and genetic modifications affecting α-syn expression, through duplications, triplications, or polymorphisms in its promoter, have been linked to both idiopathic and familial forms of PD as well as Lewy body disease. Nevertheless, neuropathological studies utilizing pan-α-syn antibodies, recognizing both physiological and pathological forms of the protein, do not consistently report a relationship between the load of Lewy body pathology and clinical disease severity. The effort in reconciling the apparent importance of α-syn in Lewy body disease, with the difficulty relating Lewy body burdens in the brain to phenotypic severity, has stimulated the search for particularly disease-relevant forms of α-syn. This protein undergoes various post-translational modifications (PTMs), including acetylation, nitration, ubiquitination and glycosylation. Phosphorylation at serine 129 (pS129) increases from approximately 4% under physiological conditions to 90% in Lewy body disease, making this modification a focus as a potential driver of the disease process.

SUMMARY

There is a need for biomarkers for Lewy body dementias, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and reagents for their detection. Aggregated non-phosphorylated α-synuclein is a biomarker for the Lewy body dementias, especially their early stages. Disclosed herein are an antibody that binds aggregated non-phosphorylated α-synuclein, and methods for generating and using such antibodies.

One aspect is a monoclonal antibody that specifically binds aggregated, non-phosphorylated α-synuclein. In some embodiments, the antibody does not bind to phosphorylated α-synuclein. In some embodiments, the antibody does not to non-phosphorylated α-synuclein monomers. In some embodiments, the epitope recognized by the antibody includes the serine residue at position 129 of α-synuclein, a site at which α-synuclein can become phosphorylated. In some embodiments, the antibody recognizes, and can be induced by, a peptide comprising or consisting of amino acid residues 125-133 of α-synuclein (SEQ ID NO:1), namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3). Such antibodies can be referred to as means for specifically binding to aggregated, non-phosphorylated α-synuclein. Some embodiments are compositions comprising a monoclonal antibody that binds aggregated, non-phosphorylated α-synuclein and a carrier, solvent, buffer, or other excipient. In some embodiments, the antibody is the monoclonal antibody produced by the hybridoma 4B1 which has been deposited with the American Type Culture Collection (ATCC), Patent Deposit Number PTA-127017. In some embodiments, the antibody has high affinity to an epitope comprising the amino acid 125-133 region with non-phosphorylated Ser at position 129 of α-synuclein. In some embodiments, the epitope is linear. In some embodiments, the antibody has high affinity for α-synuclein aggregates but low affinity for α-synuclein monomers.

One aspect is a method of generating a monoclonal antibody that binds aggregated, non-phosphorylated α-synuclein comprising immunizing a mouse with a peptide comprising or consisting of amino acid residues 125-133 of α-synuclein, namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3). The peptide consisting of the sequence CYEMPSEEGY (SEQ ID NO: 3) may be referred to as means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein. In some embodiments, the immunizing peptide is conjugated to a carrier protein, for example, keyhole limpet hemocyanin. In some embodiments, the method further comprises standard procedures for generating and selecting hybridomas, as are known to those of skill in the art. Hybridomas are produced by hybridoma technology where antibody producing B-cells are fused with myeloma cells followed by selection and screening. Monoclonal antibodies can also be produced using other technologies including phage display and single B cell antibody technology. Other embodiments are a monoclonal antibody generated by any of these methods.

One aspect is a hybridoma generated by a method comprising immunizing a mouse with a peptide comprising amino acid residues 125-133 of α-synuclein, namely YEMPSEEGY (SEQ ID NO: 2). In some embodiments, the peptide comprising amino acid residues 125-133 of α-synuclein is CYEMPSEEGY (SEQ ID NO: 3).

One aspect is a hybridoma that produces an antibody that binds aggregated, non-phosphorylated α-synuclein comprising immunizing a mouse with a peptide comprising amino acid residues 125-133 of α-synuclein, namely YEMPSEEGY (SEQ ID NO: 2). In some embodiments, the peptide comprising amino acid residues 125-133 of α-synuclein is CYEMPSEEGY (SEQ ID NO: 3).

One aspect is a hybridoma that produces an antibody that specifically binds aggregated, non-phosphorylated α-synuclein. In some embodiments, the hybridoma is 4B1, ATCC Patent Deposit Number PTA-127017. Some embodiments are antibodies having the variable region amino acid sequences or CDR amino acid sequences of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017). Some embodiments are nucleic acids (DNA or RNA) comprising encoding sequences for the variable region amino acid sequences or CDR amino acid sequences of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017).

One aspect is a method of making a monoclonal antibody that binds aggregated, non-phosphorylated α-synuclein comprising culturing a hybridoma that secretes the antibody and collecting the culture supernatant. Some embodiments further comprise purifying the antibody from the culture supernatant. In some embodiments, the hybridoma is 4B1, ATCC Patent Deposit Number PTA-127017. Other embodiments are a monoclonal antibody made by any of these methods.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-D depict the effect of pS129 on α-syn aggregation and its ability to seed the polymerization/aggregation of α-syn. FIG. 1A. Monomeric α-syn was incubated in the presence of various percentages (0, 5, 20, 50 and 100%) of monomeric pS129-α-syn (final concentration 100 μM) for 20 days at 37° C. with continuous shaking. Fibril formation was evaluated by Th-S fluorescence. The assay was performed in triplicates, and the means±standard deviations are shown. FIGS. 1B and 1C. Monomeric WT-α-syn (100 μM) was incubated alone, with 2 μM (FIG. 1B) or 0.1 μM (FIG. 1C) (final concentration) of pS129 seeds or WT seeds for 48 hours. FIGS. 1D and 1E. Samples of monomeric pS129-α-syn (100 μM) were incubated alone or with 2 μM (FIG. 1D) or 0.1 μM (FIG. 1E) (final concentration) of pS129 seeds or WT seeds for 48 hours. Fibril formation was evaluated by Th-S fluorescence. The assay was performed in triplicates, and the means±standard deviations are shown. (* p<0.05, ** p<0.01).

FIGS. 2A-G depict the effect of pS129 on nucleation-dependent polymerization by RT-QuIC assay. The assay was performed using recombinant monomeric WT-α-syn or p-S129-α-syn used as substrates in FIGS. 2A-D on samples from PD and DLB frontal and temporal regions. When used as a substrate, monomeric WT-α-syn showed higher seeding propensity compared to p-S129-α-syn as shown by shortest lag-phase (FIG. 2E), highest aggregation rate (FIG. 2F), and highest FMAX value reached at end of the reaction (FIG. 2G). The amplification curves show the mean fluorescence in each time point with standard error as shaded area. One-way ANOVA with Tukey's multiple comparison testing was used for statistical comparisons. (***, p<0.001; **, p<0.001; *, p<0.01; p<0.05).

FIGS. 3A-D depict the effect of WT- and pS129-α-syn seeding on the viability of neuroblastoma cells. The effect of WT and pS129-α-syn seeding on the viability of BE(2)-M17-WT (FIGS. 3A, 3C) and SH-SY5Y human neuroblastoma WT (FIGS. 3B, 3D) cells was estimated by the MTT assay. BE(2)-M17 (FIGS. 3A, 3C) and SH-SY5Y (FIGS. 3B, 3D) cells were treated with different concentrations of α-syn pure fibrils or pure seeds and one hour after treatment, monomeric WT or pS129-α-syn was added to a final concentration of 10 μM for 48 hours (average of 3 wells±standard deviation). The results are expressed as the percentage of the control average (i.e., untreated cells). (***, p<0.001; **, p<0.01, *, p<0.05).

FIGS. 4A-B depict the effect of pS129-α-syn seeds in seeding the aggregation of endogenously expressed α-syn-EGFP. FIG. 4A. Treatment with α-syn seeds in a concentration of 100 nM for 4 days. Representative images of cells under the different treatments. DAPI was used for nuclear staining. Scale bar=10 μm. FIG. 4B. Quantification of the percentage of cells with inclusions (>200 cells counted per condition, 4 independent experiments; mean±SD) revealed that pS129-α-syn is less potent than WT-α-syn in seeding aggregation, #: comparison WT seeds to pS129-α-syn seeds (**p<0.01).

FIGS. 5A-H depict an analysis of α-syn aggregates on organotypic slice culture model after injection of S129A-α-syn PFF. FIG. 5A. Representative images of aggregates in the DG at various time points post injection of PFF, stained for WT-α-syn with 4B1 (green) and pS129-α-syn (red). Arrowheads indicate the different types of aggregates; small LNs (right-ward pointing arrowheads in the 3 dpi panel, top and bottom arrowhead in the 5 dpi panel, downward pointing arrowhead in the 10 dpi panel, and upper right-ward pointing arrow head in 14 dpi panel), intermediate LNs (left-ward point arrowhead in 3 dpi panel, central two arrowheads in 5 dpi panel, lower two arrowheads in 7 dpi panel, upper right-ward pointing arrowheads in 10 dpi panel and bottom arrowhead in 14 dpi panel), large LNs (upper arrowhead in 7 dpi panel, lower left arrowhead in 10 dpi panes, and downward and left-ward pointing arrowheads in 14 dpi panel) and cell body inclusions (lower right arrowhead in 10 dpi panel). Scale bars=20 μm. FIG. 5B. Relative MFI of WT- vs. pS129-α-syn staining in the total population of aggregates. FIG. 5C. Area proportion of WT-α-syn in the total population of aggregates. FIG. 5D. Relative MFI of WT- vs. pS129-α-syn staining in DG aggregates. FIG. 5E. Area proportion of WT-α-syn in DG aggregates. FIG. 5F. Relative MFI of WT- vs. pS129-α-syn staining in each type of aggregate (small LNs, intermediate LNs, large LNs and cell body inclusions), all time points collapsed. FIG. 5G. Area proportion of WT-α-syn in each type of aggregate, all time points collapsed. FIG. 5H. Illustration of the changes in size and density of WT-α-syn inside aggregates, depending on their morphology. All graphs are displayed as mean±SEM, normalized to the first bar (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

FIGS. 6A-B displays immunofluorescence analysis on brain sections from mice injected with recombinant α-syn PFFs. FIG. 6A. Free-floating cryostat-cut coronal sections (30 μm) 1, 2, and 4 weeks post stereotaxic injections, covering the whole nigrostriatal axis were stained with antibodies against pS129-α-syn, WT-α-syn (4B1) and DAT. FIG. 6B. To validate whether pS129-α-syn accumulations were proteinase K (PK) resistant, sections were incubated with PK.

FIGS. 7A-C depicts expression of WT- and pS129-α-syn in post-mortem human brain tissue. FIG. 7A. Sections were obtained from the amygdala of a Parkinson's disease dementia (PDD; i-iv) and DLB (v-viii) case and stained with pS129 and 4B1 on consecutive sections. Sections were imaged at precisely the same location on both slides. Scale bars=200 μm (i, ii, v, vi), 50 μm (iii, iv, vii, viii). FIG. 7B. WT-α-syn expression in the amygdala was evaluated with disease duration across all cases, showing a strong inverse relationship. FIG. 7C. Amygdala sections from DLB and PDD cases were double-labelled with pS129 and 4B1 antibodies for immunofluorescent analysis. (Images displayed are amygdala sections from DLB).

FIGS. 8A-E depict characterization of in vitro prepared pS129-α-syn. FIG. 8A. Characterization of monomeric and aggregated α-syn by immunoblotting. FIG. 8B. Th-S fluorescence readings of the monomeric and aggregated α-syn samples. FIG. 8C. Immunoblotting for α-syn samples incubated with PLK2 at different time points. Phosphorylation at S129 was detected by pS129-α-syn-specific antibody. Total α-syn was detected by anti-α-syn (211) antibody. FIG. 8D. Fibril content estimation in the pS129-α-syn seeds, WT-α-syn seeds and aggregated α-syn by Th-S fluorescence. The assay was performed in triplicates and, the means±standard deviations are shown. FIG. 8E. Electron microscopy images of negatively stained samples of pS129-α-syn seeds, WT-α-syn seeds and aggregated α-syn (100 μM). Scale bar 500 nm.

FIGS. 9A-D depict the effect of α-syn seeding on the viability of neuroblastoma cells. The viability of BE(2)-M17 and SHSY-5Y WT human neuroblastoma cells was estimated by the MTT assay. The results are expressed as the percentage of the control average (i.e., untreated cells). BE(2)-M17 (FIG. 9A) and SHSY-5Y WT (FIG. 9B) cells were treated with different concentrations of α-syn pure fibrils or pure seeds (0.0001-10 μM) and one hour after treatment, monomeric α-syn to a final concentration of 10 μM of was added. BE(2)-M17 (FIG. 9C) and SHSY-5Y (FIG. 9D) cells were treated with 2 μM (final concentration) of α-syn pure fibrils or pure seeds and one hour after treatment, monomeric α-syn was added to a final concentration ranging between 1-20 μM. The assay was performed in triplicates and the means±standard deviation are shown.

FIGS. 10A-E depict the effect of pure seeds and fibrils on neuroblastoma cells with knocked down endogenous α-syn. The cell viability of BE(2)-M17 cells, whose endogenous α-syn has been knocked down was studied by the MTT assay. Pre-designed siRNA sequence targeting human WT-α-syn was used to silence the expression of α-syn, and non-targeting scrambled siRNA was used as negative control. FIG. 10A. Immunoblotting of cell lysates for total α-syn detection using the mouse monoclonal anti-α-syn (211) antibody and β-actin as loading control. FIG. 10B. Quantification of α-syn expression levels by densitometric analysis using ImageJ software. FIG. 10C-E. The viability of BE(2)-M17 WT cells was estimated by the MTT assay. The results are expressed as the percentage of the control average (i.e., untreated cells). BE(2)-M17 WT cells were treated with different concentrations of α-syn pure fibrils (FIG. 10C), pure seeds (FIG. 10D), or monomeric WT-α-syn (FIG. 10E). The assay was performed in triplicates and the means±standard deviation are shown. (*, p<0.05).

FIGS. 11A-B depict the effect of monomeric pS129-α-syn on the viability of neuroblastoma cells. The effect of monomeric WT- and pS129-α-syn on the viability of BE(2)-M17-WT (FIG. 11A) and SHSY-5Y human neuroblastoma WT (FIG. 11B) cells was estimated by the MTT assay. The results are expressed as the percentage of the control average (i.e., untreated cells). The assay was performed in triplicates and the means±standard deviation are shown.

FIGS. 12A-B depict the purity and specificity of 4B1 towards WT-α-syn aggregates. FIG. 12A, panel i. 50 ng of recombinant WT-, pS129-, or S129A-α-syn was loaded on 15% SDS gels and transferred to nitrocellulose membranes for western blotting. FIG. 12A, panel ii. Filter retardation assessment for 4B1 reactivity towards α-syn. F11 detects α- or β-syn, whereas the antibody E20 detects γ-syn. Filter retardation analysis of 4B1 reactivity towards human (H-α-syn) or mouse (M-α-syn). FIG. 12B, panel i. Analysis of filter retardation using monomeric- (M) or fibrillar- (F) α-syn coated on nitrocellulose membranes and detected with 4B1, Syn-1, and Syn-O2. Reactivity of 4B1 towards different α-syn aggregates including fibrils, ONE- or HNE. FIG. 12B, panel ii. 4B1 pre-incubated with monomers or ONE-oligomers and tested against pre-coated monomeric α-syn in inhibition ELISA. FIG. 12B, panel iii. Sandwich-ELISA showing the reactivity of 4B1 towards monomers or different α-syn aggregates.

FIG. 13 depicts the effect of S129A-α-syn seeding on aggregation and accumulation of insoluble pS129-α-syn in a HEK cell model. 10 μg of insoluble proteins from cell lysates of untransfected (control) and transfected HEK cells were immunoblotted proteins using antibodies specific to pS129-α-syn and total α-syn (Syn-1) at time points 6, 12, 24, 48 hours post seed transfection. Recombinant pS129-α-syn (rpS129-α-syn) and recombinant α-syn (rα-syn) proteins were loaded (50 ng) as positive controls. Re-immunoblotting with β-actin antibody was performed to normalize the amount of loaded proteins.

FIG. 14 displays a staining analysis on organotypic slice culture model at early and late time points. Illustrative images of the various types of aggregates (small LNs, intermediate LNs, large LNs and cell body inclusions) at each time point, stained for 4B1 (green) and pS129-α-syn (red). Scale bars=10 μm. The temporal developments in the structural composition of α-syn aggregates is shown with time advancing from top to bottom, and their morphological development from left to right, progressing though small and intermediate sized Lewy-neurite-like aggregates (LNs), large LNs and cell body inclusions. This figure presents representative images of these four groups of aggregates with respect to different time points, 3, 5, 7,10, 14 days post injection (dpi) to illustrate the morphological progression. As seen in the images, the predominant form in the early time points (3-5 dpi) are the small and intermediate LNs. On the other hand, at later time points (7-14 dpi), large LNs and cell body inclusions are the prevalent aggregates observed. By checking all existing aggregates, the data shows a common feature which is having a core of WT-α-syn (stained by 4B1 antibody) surrounded by pS129-α-syn (stained by anti-pS129 antibody).

DESCRIPTION

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are commonly called α-synucleinopathies and idiopathic and familial forms of these diseases have been linked to abnormal expression of α-synuclein (α-syn). Nevertheless, neuropathological studies utilizing pan-α-syn antibodies, recognizing both physiological and pathological forms of the protein, do not consistently report a relationship between the load of Lewy body pathology and clinical disease severity. However, multiple forms or α-syn arising from post-translational modifications exist. These include acetylation, nitration, ubiquitination, glycosylation, and phosphorylation. Notably, phosphorylation at serine 129 (pS129) increases from approximately 4% under normal physiology to 90% in Lewy body disease, indicating that this form of α-syn may be important in the disease process.

Some studies have associated the pS129 modification with intracellular aggregate formation leading to cell death mediated by the unfolded protein response. Rodent models have suggested that the pS129 modification exacerbates the rate of pathological protein aggregation and deposition, with subsequent negative effects on neuronal functioning. Yet other studies in animal and cellular model systems report a potentially neuroprotective function of phosphorylation. Still other studies have found the pS129 modification neither increases nor diminishes cellular toxicity and/or α-syn aggregation. Nonetheless, antibodies specific for pS129 are widely used as a putative disease relevant marker. Such studies often employ pS129-α-syn as a marker of the abundance of protein inclusions to stage disease severity and evaluate the relationship between its abundance and important clinical or pathological variables, such as disease duration, phenotypic severity or cell loss, and correlate the abundance of pS129 throughout the brain with disease severity. However, it has remained uncertain whether phosphorylation precedes protein aggregation or occurs secondarily to deposition of non-phosphorylated α-syn, or whether pS129 is a driver of pathogenicity or merely a useful marker of the neurodegenerative process. We disclose herein a monoclonal antibody specific for WT-α-syn (that is, the form that remains unphosphorylated at serine 129), useful in addressing these and other questions related to the generation of α-syn aggregates and the role of the phosphorylated and unphosphorylated forms of α-syn in aggregation propensity and cytotoxicity.

As used herein non-phosphorylated α-synuclein means α-synuclein in which the serine residue at position 129 is not phosphorylated.

The term “antibody” is herein used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An antibody broadly refers to any immunoglobulin (Ig) molecule comprised of heavy (H) chains and light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope-binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, non-limiting embodiments of which are discussed below. An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule. As used herein, the term “fragment”, when referring to an antibody should be read to mean an antigen-binding fragment. Various embodiments encompass both whole antibodies and antibody fragments, while other embodiments are limited to only whole antibodies or only antibody fragments or only one or more particular types of antibody fragment.

The term “monoclonal antibody,” as used herein, refers to an antibody obtained from identical immune cells that are clones of a unique parent cell and expressed from a particular, single encoding sequence (neglecting such variation as may arise in the expression system or cell). Typically monoclonal antibodies are monospecific in that all of the antigen binding sites contain the same complementarity determining regions (CDRs) and thus bind to the same epitope. The modifier “monoclonal” indicates the character of the antibody as being obtained from a clonal source and is not to be construed as requiring production of the antibody by any particular method. Thus the term encompasses antibodies obtained through traditional hybridoma technology, but also those obtained by phage display and other molecular cloning technologies.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen, such as that of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017). The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions termed complementarity determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively.

An “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”) refers to a molecule other than an intact or whole antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds (e.g., one or more fragments of an antibody that retain the ability to specifically bind to an antigen). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), heavy chain only antibodies (HCAb), and multispecific antibodies formed from antibody fragments. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen. However these terms may also be applied to genetically encoded fragments of the same or similar nature, in addition to those fragments produced by proteolytic digestion. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (WO 90/05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed.

The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent and/or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

The “Fab” fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain, such as those of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017). Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂ antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

“Framework” or “FR” refers to variable domain residues other than complementarity determining region (CDR) residues, such as those of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in either VH or VL sequences: FR1-CD1-FR2-CDR2-FR3-CDR3-FR4.

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

“Fv” refers to the minimum antibody fragment which contains a complete antigen-binding site, such as that of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017). In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies in humans: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. In some embodiments, recombinant technology can be used to change the isotype of an antibody.

The term “epitope” or “antigenic determinant” includes any protein or polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Different epitopes may occupy the same or topological region of an antigen. With respect to α-ayn, antibodies that bind WT-α-syn (unphosphoylated at S129), pS129-α-syn, or that bind both forms of α-syn each bind distinct epitopes.

The present disclosure also encompasses functional equivalents of the antibodies described in this specification. Functional equivalents have binding characteristics that are comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239,400; PCT Application WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424, which are incorporated in their respective entireties by reference. Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies disclosed herein, such as the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017). “Substantially the same” as applied to an amino acid sequence is defined herein as a sequence with at least about 90%, and more preferably at least about 95% sequence identity to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988) and retaining binding specificity to the target antigen. Thus some embodiments are functionally equivalent to, or substantially similar to, the amino acid sequence of the monoclonal antibody produced by the 4B1 hybridoma (ATCC Patent Deposit Number PTA-127017).

Some embodiments are antibodies comprising an antigen binding portion that is a functional equivalent of the antigen binding portion of the herein disclosed antibodies. Such antibodies are encompassed by the term “antibody means for binding WT-α-syn” or simply “means for binding WT-α-syn”.

One aspect is a monoclonal antibody that specifically binds aggregated, non-phosphorylated α-synuclein (an anti-WT-α-syn antibody). In some embodiments, the antibody does not bind to phosphorylated α-synuclein. In some embodiments, the antibody does not bind to non-phosphorylated α-synuclein monomers. In some embodiments, the epitope recognized includes the serine residue at position 129 of α-synuclein, a site at which α-synuclein becomes phosphorylated. In some embodiments, the antibody recognizes, and can be induced by, a peptide comprising or consisting of amino acid residues 125-133 of α-synuclein (SEQ ID NO:1), namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3). Such antibodies can be referred to as means for specifically binding to aggregated, non-phosphorylated α-synuclein. Some embodiments are compositions comprising a monoclonal antibody that binds aggregated, non-phosphorylated α-synuclein and a carrier, solvent, buffer, or other excipient. In some embodiments, the antibody is the monoclonal antibody produced by the hybridoma 4B1, ATCC Patent Deposit Number PTA-127017.

In various embodiments, the monoclonal antibody that specifically binds aggregated, non-phosphorylated α-synuclein does not bind one or more of pS129-α-syn, S129A-α-syn (in which serine is replaced with alanine), β-synuclein, or γ-synuclein. In various embodiments binding specificity is determined by western blot, ELISA, slot blot, or any combination thereof. In some embodiments, the antibody has high affinity to an epitope comprising amino acids 125-133 of α-synuclein with non-phosphorylated Ser at position 129 of α-synuclein. In some embodiments, the epitope is linear. In some embodiments, the antibody has high affinity for α-synuclein aggregates but low affinity for α-synuclein monomers. In a facet of these embodiments, the α-synuclein aggregates are protofibrils or soluble oligomers of α-synuclein and the antibody can have high affinity for the protofibrils, the soluble oligomers, or both. In other facets of these embodiments, the α-synuclein aggregates are α-synuclein fibrils.

One aspect is a method of generating an antibody that binds, or antiserum specific for, aggregated, non-phosphorylated α-synuclein comprising immunizing a mouse or other laboratory animal (for example, a rat, a hamster, or a rabbit) or an agricultural animal (for example, a goat, a sheep, or a horse) with a peptide comprising of amino acid residues 125-133 of α-synuclein (SEQ ID NO:1), namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3) may be referred to as means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein. In some embodiments, the immunizing peptide is conjugated to a carrier protein, for example, keyhole limpet hemocyanin, Concholepas concholepas hemocyanin, bovine serum albumin, ovalbumin, or diphtheria or tetanus toxoid. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the method further comprises standard procedures for generating and selecting hybridomas, as are known to those of skill in the art. Some embodiments are a monoclonal antibody generated by any of these methods.

One aspect is an antibody that binds, or antiserum specific for, aggregated, non-phosphorylated α-synuclein made by a process comprising immunizing a mouse with a peptide comprising or consisting of amino acid residues 125-133 of α-synuclein (SEQ ID NO: 1), namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3). Some embodiments further comprise applying hybridoma technology or molecular cloning technology to obtain a monoclonal antibody.

Some embodiments are a monoclonal antibody generated by (or made by a process comprising) immunizing a laboratory animal with means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein. In some embodiments, the laboratory animal is a mouse. In some embodiments, the means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein are conjugated to a carrier protein. In some embodiments, the carrier protein is keyhole limpet hemocyanin. In some embodiments, the means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein is the peptide CYEMPSEEGY (SEQ ID NO:3). Some embodiments further comprise screening hybridomas for reactivity of the antibody against recombinant WT-α-syn. Some embodiments further comprise a counter-screens for a lack of reactivity of the antibody against one or more of pS129-α-syn, S129A-α-syn (in which serine is replaced with alanine), β-synuclein, or γ-synuclein. In some embodiments, the screening (or counter-screening) comprises an ELISA. In some embodiments, the screening (or counter-screening) comprises filter retardation assay analysis.

With respect to any of the antibody aspects, some embodiments are pharmaceutical compositions comprising the antibody. A pharmaceutical composition is one intended and suitable for the treatment of disease in humans. That is, it provides overall beneficial effect and does not contain amounts of ingredients or contaminants that cause toxic or other undesirable effects unrelated to the provision of the beneficial effect. A pharmaceutical composition will contain one or more active agents and may further contain solvents, buffers, diluents, carriers, and other excipients to aid the administration, solubility, absorption or bioavailability, and or stability, etc. of the active agent(s) or overall composition. A “pharmaceutically acceptable carrier, diluent, or excipient” is a medium generally accepted in the art for the delivery of biologically active agents to mammals, e.g., humans. The compounds of the present invention can be formulated as pharmaceutical compositions using a pharmaceutically acceptable carrier, diluent, or excipient and administered by a variety of routes. In particular embodiments, such compositions are for oral or intravenous administration. Such pharmaceutical compositions and processes for preparing them are well known in the art. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro, et al., eds., 19. sup. th ed., Mack Publishing Co., 1995).

Aspects of the present specification provide, in part, administering a therapeutically or prophylactically effective amount of an anti-α-synuclein antibody or a pharmaceutical composition thereof. As used herein, the term “therapeutically effective amount” is synonymous with “therapeutically effective dose” and when used in reference to treating a neurodegenerative disorder associated with α-synuclein, such as Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease, or multiple system atrophy, means at least the minimum dose of a compound or composition disclosed herein necessary to achieve the desired therapeutic or prophylactic effect. In some embodiments, it refers to an amount sufficient to prevent, slow, or halt the neurodegenerative process. In some embodiments, it includes a dose sufficient to reduce a symptom associated with the neurodegenerative disease. An effective dosage or amount of an anti-α-synuclein antibody or a composition thereof can readily be determined by the person of ordinary skill in the art considering all criteria (for example, the rate of excretion of the compound or composition used, the pharmacodynamics of the compound or composition used, the nature of the other compounds to be included in the composition, the particular route of administration, the particular characteristics, history and risk factors of the individual, such as, e.g., age, weight, general health and the like, the response of the individual to the treatment, or any combination thereof) and utilizing his best judgment on the individual's behalf.

The terms “treatment,” “treating”, etc., refer to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. Various embodiments may specifically include or exclude one or more of these modes of treatment. The herein disclosed anti-α-synuclein antibodies may be used as medicaments for the treatment of α-synuclein-associated neurodegenerative disorders. Further embodiments are methods of treating α-synuclein-associated neurodegenerative disorders comprising administering an anti-α-synuclein antibody to a subject in need thereof. In some embodiments, the α-synuclein-associated neurodegenerative disorder is Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease, or multiple system atrophy. In some embodiments, the subject in need thereof is a human.

One aspect is a method of making a monoclonal antibody that binds aggregated, non-phosphorylated α-synuclein comprising culturing a hybridoma that secretes the antibody and collecting the culture supernatant. Some embodiments further comprise purifying the antibody from the culture supernatant. The antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, proteins G-Sepharose, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt).

In some embodiments, the hybridoma is 4B1, ATCC Patent Deposit Number PTA-127017. Further embodiments are a monoclonal antibody that binds aggregated, non-phosphorylated α-synuclein made by any of these method of making embodiments.

Various aspects are a hybridoma that produces an antibody that specifically binds aggregated, non-phosphorylated α-synuclein. In some embodiments, the hybridoma is 4B1, ATCC Patent Deposit Number PTA-127017. One aspect is a hybridoma generated by a method comprising immunizing a mouse with a peptide comprising or consisting of amino acid residues 125-133 of α-synuclein (SEQ ID NO: 1), namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3). One aspect is a hybridoma that produces an antibody that binds aggregated, non-phosphorylated α-synuclein comprising immunizing a mouse with a peptide comprising or consisting of amino acid residues 125-133 of α-synuclein, namely YEMPSEEGY (SEQ ID NO: 2), for example, the peptide CYEMPSEEGY (SEQ ID NO: 3).

One aspect is an immunoassay utilizing an anti-WT-α-syn antibody to detect or quantitate WT-α-syn. An exemplary, non-limiting immunoassay is an enzyme-linked immunosorbent assay (ELISA). In other embodiments, the immunoassay is an immunohistochemical assay. Immunoassays measure substances, such as analytes, proteins, etc., using the specificity of an antibody to the substance. In some embodiments, the anti-WT-α-syn antibody is the antibody produced by the hybridoma is 4B1, ATCC Patent Deposit Number PTA-127017. In some embodiments, the immunoassay may further utilize an antibody specific for pS129-α-syn.

One aspect is a sandwich ELISA. In such an assay, a capture antibody specific for the substance is associated with a solid support, such as a microtiter plate. A liquid containing the substance (or suspected of containing the substance, or a sample in need of determining not to include the substance) is allowed to bind to the capture antibody. Then a detection antibody, also specific for the substance, is added to allow detection of substance bound to the capture antibody. In some embodiments, the anti-WT-α-syn antibody is coated on the surface of a microtiter well or bead and used as the capture reagent and a pan-α-syn antibody is used as the detection reagent. In other embodiments, a pan-α-syn antibody is used as the capture reagent and the anti-WT-α-syn antibody is used as the detection reagent. In some embodiments, the pan-α-syn antibody is 11D12 (Majbour et al., Mol Neurodegener. 11, 7, 2016)

Another aspect is an immunohistochemical assay. Immunohistochemistry involves the process of selectively imaging antigens (proteins) in a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, a detection antibody is used which allows the detection of the anti-α-synuclein antibody and thus the substance to which the antibody is bound.

In some embodiments, the detection antibody is a labeled antibody. In other embodiments, the anti-α-synuclein antibody is labeled. The label can include a radioactive label, an enzyme label, a colorimetric label, a fluorescent label, a chemiluminescent label, or other labels known to persons of skill in the art. In some such embodiments, the detection antibody is biotinylated so that it can bind an enzyme-linked avidin molecule, such as streptavidin conjugated with horseradish peroxidase or alkaline phosphatase. In alternative embodiments, avidin molecule is conjugated with another detectable label, for example, a fluorescent dye or quantum dot. In some embodiments, the detection antibody is directly labeled. Still further alternatives are familiar to one of skill in the art.

In further embodiments in which the anti-α-synuclein antibody is labeled, the anti-α-synuclein antibody is used for in vivo imaging by administering the labeled anti-α-synuclein antibody to a subject and detecting the label.

In some embodiments, the label is an enzymatic label such as a peroxidase (e.g., horseradish peroxidase), a galactosidase (e.g., β-D-galactosidase), or a phosphatase (e.g., alkaline phosphatase). For enzymatic labels, a substrate is needed which is cleaved by the enzyme to produce a color, fluorescence, or luminescence, which is measured spectrophotometrically. Exemplary colorimetric substrates for peroxidase include, but are not limited to, 3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′,4,4′ diaminobenzidine (DAB), 4-chloro-1-naphthol (4CN), 2,2′-azino-di [3-ethylbenzthiazoline] sulfonate (ABTS), and o-phenylenediamine (OPD). In some embodiments, when the assay is an ELISA, the substrate is TMB which produces a blue color which is measured at a wavelength of 650 nm. The reaction can be halted by addition of acid or another stop reagent. Using a sulfuric acid stop solution turns TMB yellow and the color can then be read at 450 nm. Exemplary colorimetric substrates for phosphatase include, but are not limited to, 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (BCIP/NBT) and p-nitrophenylphosphate (p-NPP). Exemplary colorimetric substrates for galactosidase include, but are not limited to, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl), and β-D-galactopyrenoside (DDAO galactoside). Exemplary fluorescent substrates include, but are not limited to, 4-methylumbelliferyl phosphate (4-MUP; for phosphatase), and 4-methylumbelliferyl galactoside (MUG; for galactosidase), fluorescein di-β-D-galactopyranoside (FDG; for galactosidase), hydroxyphenylacetic acid (HPA; for peroxidase), and 3-p-hydroxyphenylproprionic acid (HPPA; for peroxidase). Exemplary luminescent substrates include, but are not limited to, luminol, polyphenols (e.g., pyrogallol, pupurogallin, gallic acid, and umbelliferone) and acridine esters, and luciferin for peroxidase; 3-(2′-spiroadamantane)-4-methyl-4-(3′-phosphoryloxyphenyl-1, 2-dioxetane, disodium salt) (AMPPD) for phosphatase; and (3-(2′-spiroadamantane)-4-methoxy-4-(3′-β-D-galactopyranosyloxyphenyl-1,2-dioxetane (AMPGD) for galactosidase.

In some embodiments, the label is horseradish peroxidase and the substrate is TMB.

In some embodiments, the label is a colorimetric label, a fluorescent label, or a luminescent label. An exemplary colorimetric label includes, but is not limited to, nanoparticulate gold. Exemplary fluorescent labels include, but are not limited to, ethidium bromide, fluorescein and its derivatives, rhodamine and its derivatives, green fluorescent protein, Texas Red, Cascade Blue, Oregon Green, Marina Blue, an atto label, a CF™ dye, an Alexa Fluor, and a cyanine dye. Exemplary luminescent labels include, but are not limited to, luciferin and firefly luciferase.

With respect to the assay aspects, some embodiments the assay is used as a diagnostic assay to determine whether or not an individual has a neurodegenerative disease associated with α-synuclein, for example, Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease, or multiple system atrophy. In some embodiments, the diagnostic assay comprises adding the antibody to a biological sample from a subject, and detecting the presence or absence of a complex formed between α-synuclein aggregates and the antibody or fragment.

With respect to the assay aspects, some embodiments comprise a test kit comprising an anti-α-synuclein antibody and other reagents or equipment needed to carry out the assay.

EXAMPLES

The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples should not be construed to limit any of the embodiments described in the present specification,

Example 1 Generation of Monoclonal Antibody Recognizing Non-Phosphorylated S129-α-Synuclein (WT-α-syn) Aggregates

A short synthetic peptide, CYEMPSEEGY (SEQ ID NO:3), designed over the region of interest (amino acids 125-133 of α-syn; YEMPSEEGY (SEQ ID NO:2)), was used as the immunogen. This peptide was solubilized in phosphate-buffered saline (PBS) and conjugated to keyhole limpet hemocyanin (KLH) as carrier protein. Experimental procedures using mice were carried out in accordance with Laboratory Animal Research Center (LARC), Qatar University (QU), Qatar, according to the QU institutional ethical rules and regulations and approved by QU—IACUC & IBC. Specifically, female BALB/c mice (6-8 weeks old) were injected subcutaneously with the immunogen conjugate. Ten days post booster immunization, blood was collected from the tail vain and titer response was evaluated using indirect ELISA. Mice exhibiting a strong immune response were subjected to a final immunization before euthanization. Following hybridoma generation, monoclonal antibodies specific for WT-α-syn were obtained. Culture supernatants were tested for secreted anti-α-syn antibodies using indirect ELISA.

For the indirect ELISA, a 96-well clear plate was coated with recombinant WT-α-syn and incubated overnight at 4° C. The following day, the plate was blocked with blocking buffer (PBST containing 2.25% gelatin) for 1 hour at RT. The plate was then washed three times with PBST, and antisera from the mice, or culture supernatants, were added in serial dilutions. Next, the plate was washed and goat anti-mouse IgG-HRP (1:20 K, Jackson ImmunoResearch) was added. The plate was washed again and detected with TMB substrate (Abcam). Following color development, the reaction was stopped by addition of 0.6 N H₂SO₄ and the absorbance was measured at 450 nm.

To generate hybridoma, splenocytes were fused with mouse myeloma cells (Sp2O-Ag14; American Type Culture Collection) at a ratio of 5:1 and fusion was induced using 50% polyethylene glycol. Fused cells were seeded in 96-well plate in IMDM media (Gibco) containing HAT (Sigma). Positive clones were transferred to 24-well plates and screened multiple times to ensure stability.

For isotyping, culture supernatant was screened against anti-mouse heavy chain antibodies (Isotyping Kit, Sigma-Aldrich) in ELISA and only IgG-positive clones were selected. Those were subjected to single-cell cloning. Wells with single clones were grown to confluency and screened at least three times for further selection of stable clones. Selected clones were grown in CDM4mAb media (Hyclone) to confluency. Culture supernatant was then collected and purified using protein-G agarose affinity chromatography (Sigma-Aldrich). Several monoclonal antibodies were produced, purified and thoroughly characterized. An IgG1 monoclonal antibody designated 4B1 was selected for further characterization.

The purity of 4B1 antibody was assessed using SDS-PAGE under reducing conditions. The specificity of 4B1 for recombinant WT-, pS129-, or mutated S129A-α-syn (with a substitution of serine 129 to alanine) proteins was assessed by western blot. The data showed that 4B1 is specific for WT-α-syn and did not recognize pS129-α-syn (FIG. 12A panel i). Furthermore, there was no evident band when serine was replaced with alanine (S129A-α-syn) indicating that S129 is an integral residue of 4B1's epitope (FIG. 12A, panel i). Syn-1 (mouse anti-α-syn, BD Bioscience) and PS129 (in-house mouse anti pS129-α-syn antibody (Majbour et al., Mol Neurodegener. 11(7), 2016), antibodies were included as controls (FIG. 12A, panel i). Filter retardation assay analysis showed that 4B1 specially recognized α-syn sparing β- and γ-syn (FIG. 12A, panel ii). F11 (mouse monoclonal anti-α/β/γ synuclein antibody, Santa Cruz Biotechnology), E-20 (mouse monoclonal anti-γ-syn antibody, Santa Cruz Biotechnology) were included as control antibodies. 4B1 recognized both human and mouse α-syn to equal extent (FIG. 12A, panel ii). Additionally, 4B1 reacted equally to WT-α-syn from human or mouse species.

Hybridoma 4B1 was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas VA, 20110-2209 on Jun. 24, 2021 and given the Patent Deposit Number (accession number) PTA-127017.

Example 2 Specificity of 4B1 Towards WT-α-syn Aggregates

Using filter retardation assay, an assay that preserves the protein conformation, surprisingly we found that 4B1 only recognized WT-α-syn aggregates, and did not recognize the monomeric form of α-syn protein (FIG. 12Bi). Syn-O2 (a mouse monoclonal antibody against α-syn aggregates (Vaikath et al., Neurobiol Dis. 79:81-99, 2015)), and Syn-1 were used as control antibodies (FIG. 12B, panel i).

Given that α-syn undergoes different conformational changes forming oligomers and amyloid fibrils, we further assessed the specificity of 4B1 antibody against different conformations of the protein. α-Syn oligomers were prepared in vitro either through spontaneous formation during α-syn fibrillization, or in the presence of a cross-linking reagent such as the lipid peroxidation products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal(HNE). Both oligomers hold distinct compositions and conformations (Uversky, Cell Mol Life Sci. 60(9):1852-1871, 2003; Pieri et al., Sci Rep. 6(24526), 2016). 4B1 showed higher affinity towards beta-sheet rich aggregates (fibrils, ONE-, or HNE-oligomers) (FIG. 12B, panel i). To further evaluate the selectivity and affinity of 4B1 antibody towards α-syn aggregates we performed an inhibition ELISA. Our data showed that 4B1 antibody selectively recognized oligomers compared to monomers (FIG. 12Bii). 4B1 was also tested against serial dilutions of α-syn monomers, ONE- and HNE- oligomers, and pS129-α-syn aggregates using sandwich-based ELISA assay. Affirming previous observations, 4B1 specifically recognized α-syn oligomers sparing both the monomeric and phosphorylated forms (FIG. 12B, panel iii). Altogether, the data strongly suggests that 4B1 is specific for beta-sheet rich aggregates of WT-α-syn.

Example 3 pS129 α-syn Inhibits α-syn Aggregation

Knowing that under physiological conditions only 4% of α-syn is phosphorylated at S129, we investigated the effect of pS129-α-syn on α-syn aggregation. Pure monomeric α-syn was mixed with different concentrations of pS129-α-syn (0-100%). Samples were incubated up to 20 days, and Th-S binding assay showed the aggregation at various time points. As expected, the non-phosphorylated monomeric α-syn aggregated gradually reaching approximately 35,000 Th-S counts after 20 days of incubation (FIG. 1A). The sample containing 5% pS129-α-syn exhibited a similar aggregation trend to the non-phosphorylated sample (FIG. 1A). Surprisingly, the sample with 20% of pS129-α-syn showed a significant decrease in aggregation, given that the reduction in Th-S fluorescence readings was more than 50%. Moreover, the fibril formation in the samples containing 50% and 100% of pS129-α-syn was almost depleted, with Th-S fluorescence readings reaching approximately 2,000 counts after 20 days of incubation. The results indicate that phosphorylation at S129 exerts an inhibitory effect on α-syn aggregation.

Example 4 pS129 α-syn has Reduced Ability to Seed the Aggregation of α-syn

We further assessed whether pS129-α-syn had an effect on seeding α-syn aggregation. For this purpose, in vitro phosphorylated monomeric and aggregated recombinant α-syn (characterized by immunoblotting and Th-S binding (FIG. 8A, 8B) were utilized. The phosphorylation of α-syn was confirmed by immunoblotting with a pS129-α-syn specific antibody (FIG. 8C). WT and pS129-α-syn pure seeds were characterized by Th-S fluorescence and EM (FIG. 8D, 8E). Upon addition of WT-α-syn seeds (2 μM), monomeric α-syn aggregation was dramatically accelerated as shown by increasing Th-S fluorescence readings throughout the incubation period compared to monomeric α-syn alone (FIG. 1B). It is worth noting that WT-α-syn seeds (2 μM) had a greater impact on the aggregation of monomeric α-syn compared to pS129-α-syn seeds (FIG. 1B). However, upon addition of seeds at lower concentration (0.1 μM), pS129-α-syn seeds completely failed to aggregate WT-α-syn monomers, whereas, WT-α-syn seeds at the same concentration significantly accelerated the monomeric α-syn aggregation (FIG. 1C). Moreover, we studied the seeding effect of pS129-α-syn monomers. The data illustrated that pS129-α-syn monomers failed to aggregate even after 48 hours of incubation after adding either WT-α-syn seeds or pS129-α-syn seeds, indicating that phosphorylation at S129 had an inhibitory effect on α-syn aggregation (FIG. 1D, E).

Example 5 Inhibitory Seeding Effect of pS129-α-syn on RT-QuIC Assay

Next, we evaluated the impact of phosphorylation at S129 on the seeded nucleation-dependent polymerization assay (RT-QuIC) for α-syn (Fairfoul et al., Ann Clin Transl Neurol. 3(10):812-818, 2016). This assay was carried out in the temporal and frontal cortex samples extracted from four PD and four DLB cases. We performed the assay using WT- and pS129-α-syn monomers. Interestingly, as shown in FIG. 2 , monomeric WT-α-syn showed higher seeding propensity compared to pS129-α-syn that remained negative with the unseeded reaction (FIG. 2A-H). This data confirmed the inhibitory effect of pS129 modification on α-syn aggregation. α-Syn seeding activity was detected in all the cases where WT-α-syn was used as substrate (FIG. 2A-H). To better characterize the RT-QuIC efficiency and simplify the results for comparison, we assessed the RT-QuIC kinetic parameters of the reactions seeded with brain homogenates. When compared to pS129-α-syn, WT-α-syn showed the shortest lag-times (FIG. 2F, Table 1), the highest amyloid formation rates (FIG. 2G) and the highest FMAX (FIG. 2H).

TABLE 1 RT-QuIC kinetic parameters of the relative seeding activity for monomeric α-syn/pS129-α-syn in frontal and temporal regions extracted from PD and DLB cases. Amyloid formation F_(MAX) rate ± (RFU × lag-phase ± st dev 10³) ± Seed Substrate stdev (1/hours) stdev PD α-syn  26 ± 3.4 0.04 ± 0.005 180 ± 57 Frontal pS129-α-syn  94 ± 45 0.013 ± 0.008   69.8 ± 58.7 PD α-syn 19.8 ± 2.5 0.05 ± 0.006  199 ± 34.8 Temporal pS129-α-syn 104 ± 34 0.01 ± 0.003 32.2 ± 5  DLB α-syn 37.8 ± 6  0.027 ± 0.005  131 ± 93 Frontal pS129-α-syn 132 ± 31 0.007 ± 0.0019 27.7 ± 4  DLB α-syn 22.5 ± 4  0.045 ± 0.008    259 ± 0.571 Temporal pS129-α-syn  88 ± 45 0.013 ± 0.006  118 ± 99

Example 6 Addition of pS129-α-syn Monomers to Preformed Aggregates in Cells did not Promote Seeding-Mediated Toxicity

The cytotoxicity effect of pure seeds and fibrils (2, 5 and 10 μM) on BE(2)-M17 and SH-SY5Y WT cells in the presence or absence of WT-α-syn monomers was evaluated. Both α-syn species affected cell viability (both BE(2)-M17 and SH-SY5Y) at all tested concentrations as shown in FIGS. 3A and B. Despite the fact that seeds exhibited more toxicity than fibrils, adding monomeric WT-α-syn significantly exacerbated this effect. Given that the effect was independent of pure seeds/fibrils concentration, cell viability was evaluated upon varying both the concentration of seeds and fibrils (lower concentration scale, of 0.0001-10 μM) in the presence of a constant concentration of α-syn monomers and vice versa (FIG. 9 ). As shown in FIG. 9A, 9B, pure seeds and fibrils decrease cell viability in a concentration-dependent fashion, with a more pronounced toxic effect in the presence of monomeric α-syn (final concentration of 10 μM), with seeds being more toxic than fibrils. Additionally, different concentrations of monomeric α-syn (1-20 μM) on cell viability were assessed, with cells first treated with a constant concentration (2 μM) of either pure fibrils or seeds. As indicated in FIG. 9C, 9D, the addition of monomers seems necessary for cell toxicity when pre-treated with pure fibrils or seeds since α-syn monomers alone had no effect.

To further emphasize the role of monomeric α-syn in inducing the nucleation polymerization process of α-syn, the effect of pure seeds and fibrils on the viability of BE(2)-M17 WT cells with siRNA silenced endogenous α-syn was also examined. Immunoblotting and its corresponding quantification of α-syn expression are shown in FIG. 10A, 10B. The results demonstrated that the cells whose endogenous α-syn was knocked down were less susceptible to the toxic effects of pure fibrils (FIG. 10C) and seeds (FIG. 10D). However, the treatment of cells with monomeric α-syn at all given concentrations had similar effects in siRNA-transfected and control cells (FIG. 10E). The data confirm the important role of monomeric α-syn in aggregation-induced toxicity.

Our findings prompted us to study the effect of monomeric pS129-α-syn alone compared to WT-α-syn on the toxicity of both BE(2)-M17 and SH-SY5Y WT cells. Monomeric pS129-α-syn employed at a range of concentrations (0.31-20 μM) did not show toxicity in BE(2)-M17 cells (FIG. 11A) and SH-SY5Y cells (FIG. 11B). For this purpose, adding monomeric pS129-α-syn to cells pre-treated with either pure fibrils or pure seeds was assessed for toxicity. BE(2)-M17 and SH-SY5Y WT cells were treated with different concentrations of either pure fibrils or pure seeds (10, 5 and 2 μM) and after one hour of incubation, monomeric pS129-α-syn was added to the cells at a final concentration of 10 μM. As shown in FIG. 3C, 3D, pS129 monomers had no effect on viability of the cells pre-treated with various concentrations of pure fibrils or seeds. The toxicity levels observed were comparable and statistically non-significant to the group treated only with pure seeds or fibrils.

Example 7 pS129-α-syn Seeds is Less Potent in Seeding α-syn Aggregation in Cells

To further investigate the seeding efficiency of α-syn seeds in vitro, we used a stable cell line over-expressing α-syn fused to EGFP (HEK293-aSyn-EGFP) under the CMV promoter, to examine whether the exogenous addition of the different α-syn seeds affected the seeding of endogenous α-syn. We treated cells with either WT-α-syn seeds or pS129-α-syn seeds at a final concentration of 100 nM and incubated for four days. Control cells were exposed to vehicle only (PBS). Cells were then processed for microscopy and the percentage of cells with intracellular accumulation of α-syn-EGFP was counted. Interestingly, we found that the internalized WT-α-syn seeds resulted in a significant increase of the α-syn-EGFP inclusions, compared to control cells (FIG. 4A). In contrast, exposure of cells to pS129-α-syn seeds caused a significantly lower percentage of cells with α-syn inclusions when compared to the WT-α-syn-treated cells, but yet significantly higher than control cells (FIG. 4B).

Example 8 WT-α-syn is Predominant at Early Stages in a Seeding Dependent Aggregation In Vitro Model of HEK Cells

Given the central role of seeding in inducing α-syn fibril formation, seeding-dependent aggregation by mutant S129A-α-syn seeds, and the formation of insoluble pS129-α-syn was studied in an in-vitro cell model consisting of α-syn expressing HEK cells. Insoluble pS129-α-syn was induced after a consecutive transfection of WT-α-syn and mutant S129A-α-syn seeds. These results indicate that pS129-α-syn is generated at later stages especially at 24 and 48 hours post seed treatment (FIG. 13 ). On the contrary, insoluble WT-α-syn detected by 4B1, showed a gradual decrease in its expression with the greatest effect at 48 hours post transfection (FIG. 13 ). These findings confirm that the levels of the two forms of α-syn protein are inversely related, with insoluble WT-α-syn having its highest formation at early stages and pS129-α-syn appearing at later stages.

Example 9 Slice Culture Model Displays a Maturation of Aggregates With Increasing Phosphorylation at S129 Over Time

In order to investigate any temporal developments in the structural composition of α-syn aggregates, we employed a recently described organotypic slice culture model where α-syn aggregation is induced by injection of S129A-α-syn PFF (Elfarash et al., Acta Neuropathol Commun. 7(213), 2019). Evaluating slices at both early and later time points after induction of aggregation and stained by 4B1 revealed a continuous decrease in both the WT-α-syn -stained proportion of aggregates and in the relative mean fluorescence intensity (MFI) of the WT-α-syn aggregate staining compared with the pS129-α-syn staining (FIG. 5A-C). The same pattern was visible when focusing the analysis on the dentate gyrus (DG), the region in which aggregate formation is induced and is thus by far most abundant (FIG. 5D-E). The decrease in both area proportion and MFI of WT-α-syn (representing the density of staining) effectively highlights an increase in the phosphorylation level of α-syn aggregates over time, indicating maturation of the aggregates taking place.

To further investigate this maturation, analysis was stratified against morphology of aggregates, dividing them into four groups. At early time points (3-5 days post injection (dpi)), the predominant aggregate types are small and intermediate sized Lewy-neurite-like aggregates (LNs), while at later time points (7-14 dpi) large LNs and cell body inclusions start appearing. Thus, the changes in WT-α-syn area proportion and relative MFI might reflect differences between the various types of aggregates. Examining the individual aggregate types showed that all aggregate types possessed a core of WT-α-syn, which was surrounded by pS129-α-syn (FIG. 14 ). Moreover, the relative WT-α-syn MFI of the small LNs was much higher than that of any other type of aggregate (FIG. 5F). Conversely, the area proportion of WT-α-syn in the aggregates was relatively stable between small LNs, intermediate LNs and cell body inclusions, while large LNs displayed a significantly smaller core of WT-α-syn (FIG. 5G). The results thus indicate a maturation process of the aggregates, where small LNs possess a large, dense core of WT-α-syn. In the intermediate LNs, the density of the WT-α-syn core is decreased, but the relative size of the core is unchanged, in contrast with the large LNs, where the core size is decreased while the density of WT-α-syn stays fixed (FIG. 5H). Curiously, cell body inclusions appear to have a larger WT-α-syn core than the large LNs, similar to the intermediate LNs.

Example 10 pS129-α-syn Occurs at a Late Stage in Mice Injected With α-syn Preformed Fibrils

Immunohistochemical analysis of striatal sections from mice injected with PFFs showed accumulation of WT-α-syn at the early stages, mainly at one and two weeks post injection of PFFs (FIG. 6A). On the contrary, evident formation of pS129-α-syn was not apparent until 4 weeks post injection (FIG. 6A), indicating its occurrence at late phases. For further validation, striatum sections were also subjected to PK treatment. These sections showed that WT-α-syn accumulations were PK resistant as shown at two weeks post injection (FIG. 6B).

Example 11 WT-α-syn is Found in the Center of Most Lewy Pathological Lesions, and its Load Correlates Inversely With Disease Duration

Immunohistochemical staining on PDD and DLB post-mortem human brain tissue demonstrated that WT-α-syn is found in Lewy neurites and Lewy bodies, though its load is approximately four-fold lower than total α-syn labelled by a pan-α-syn antibody (FIG. 7A). When normalized to total α-syn levels, there was no significant difference in load of either WT- or pS129-α-syn between Parkinson's disease dementia (PDD)and DLB cases, though WT-α-syn expression in the amygdala had a strong inverse relationship with disease duration across all cases (FIG. 7B). Using a Leica SP8 confocal microscope we performed z-stack image acquisition and 3D reconstruction using LasX software to determine the relative localization of non-phosphorylated S129 compared to pS129 α-syn. Using this method, we determined that the majority of α-synuclein accumulations consisted of a non-phosphorylated S129 core surrounded by pS129. Remarkably, confocal microscopy of PDD and DLB cases revealed a consistent picture of the core of most aggregates containing WT-α-syn, surrounded by a corona of pS129-α-syn (FIG. 7C). Overall, these findings support the suggestion that pS129 occurs secondarily to α-synuclein aggregation.

Example 12 Experimental Methods

Expression and Purification of Recombinant Human α-syn

Full-length recombinant human α-syn was expressed in Escherichia coli BL21 (DE3) using the bacterial expression vector pRK172 (Vaikath et al., Neuropathol Appl Neurobiol. 45(6): 597-608, 2019). Following expression and sedimentation, the bacterial pellets from 1 liter of Terrific broth (TB) were homogenized and sonicated in 50 ml of high-salt buffer (0.75 M NaCl, 10 mM Tris, pH 7.6, 1 mM EDTA) containing a cocktail of protease inhibitors (Thermo Scientific), heated to 100° C. for 10 min, and centrifuged at 5300 g for 20 min. The solution was dialyzed overnight against the buffer used for gel filtration chromatography (50 mM NaCl, 10 mM Tris, pH 7.6, 1 mM EDTA), following which, the volume was reduced to 5 ml using a Pierce protein concentrator (10K MWCO; ThermoFisher Scientific) according to the manufacturer's instructions. All proteins were purified by size exclusion using a Superdex 200 gel filtration column (GE Healthcare). The clean fractions were pooled, exchanged with a buffer (10 mM Tris pH 7.6, 25 mM NaCl, 1 mM EDTA, 1 mM PMSF) for ion exchange chromatography by dialysis overnight, and were applied onto a HiTrap Q column (GE Healthcare) and eluted in 10 mM Tris pH 7.6 using a linear gradient of 0.025-1.0 M NaCl. For preparation of α-syn monomers, the protein went through 100 KDa filters to remove any high molecular weight proteins. Purified fractions were pooled, and protein concentrations were determined using the Pierce BCA protein assay kit (ThermoFisher Scientific).

In Vitro Phosphorylation of α-syn

Purified α-syn was phosphorylated at S129 as described previously (Landeck et al., Mol Neurodegener. 11(61), 2016). Briefly, 1 μg of Polo-like kinase 2 (PLK2) protein was added to 1.44 mg/ml (100 μM) α-syn in kinase reaction buffer (20 mM HEPES, 1.09 mM ATP, 2 mM DTT, 10 mM MgCl₂, pH 7.4). The reaction mixture was incubated at 30° C. for 24 h.

Aggregation of α-syn In Vitro

The purity of α-syn was >95% as estimated by SDS gels. The α-syn samples were placed in 1.5 ml sterile polypropylene tubes and sealed with parafilm, followed by incubation at 37° C. for several days with continuous shaking at 800 rpm in a Thermomixer (Eppendorf). The samples were collected at the indicated time points, while the aggregation of α-syn was monitored by Th-S binding assay. The samples were stored at −80° C. for future analyses. Recombinant monomeric α-syn was mixed with various percentages of in vitro prepared monomeric pS129-α-syn (100, 50, 20, 5 or 0%) in 1.5 ml sterile polypropylene tubes, followed by incubation for up to twenty days.

Thioflavin-S (Th-S) Assay

A Th-S binding assay was used to study α-syn fibril formation. Being a fluorescent dye, Th-S interacts with fibrils containing β-sheet structures. The sample (10 μl) was diluted in 40 μl of Th-S (20 μM) in PBS and the mixture was dispensed in a 384-well, untreated black microplate (Nunc). Fluorescence was measured in a microplate reader (Perkin Elmer Envision) with the excitation and emission wavelengths at 450 and 510 nm, respectively.

Preparation of α-syn Pure Fibrils and Pure Seeds

Monomeric α-syn (100 μM) was aggregated as described above for 7 days. For preparation of pure fibrils, the crude α-syn fibril sample was spun at 10,000 xg for 10 min at 4° C. in a refrigerated microfuge (Eppendorf). The supernatant was then discarded, and the pellet was washed twice and finally resuspended in 1XPBS. For preparation of pure seeds, the pure fibrils were fragmented by ultrasonication while kept on ice using a Sonic ruptor 250, equipped with a fine tip (2 second pulses, output. of 40 watts for 5 min). For measurement of α-syn concentration of both fibrils and seeds, the samples were denatured with equal volume of 6 M Guanidine-HCl and quantified using Pierce BCA protein assay kit (ThermoFisher Scientific).

Seeded Polymerization Assay

The aggregation of monomeric WT- or pS129-α-syn with or without addition of seeds was conducted as previously described (Di Giovanni et al., J Biol Chem. 285(20):14941-14954, 2010). The seeds (WT or pS129) were prepared by fragmenting mature α-syn fibrils via sonication. Monomeric α-syn (100 μM) was seeded with different seed concentrations under incubation at 37° C. with continuous shaking. The fibril formation was assessed by the Th-S binding assay.

Isolation of TBS-Soluble Fraction from Brain Tissues

Brain tissues derived from the temporal and frontal cortex were homogenized on ice with a glass tissue homogenizer at 10% (w/v) in TBS (20 mM Tris-HCl pH 7.4, 150 mM NaCl) and 5 mM EDTA with protease and phosphatase inhibitors (ThermoFisher Scientific). Samples were centrifuged at 3000 xg, at 4° C. for 30 min. The collected supernatant represents the TBS-soluble fraction. The total protein concentration was measured by BCA assay (ThermoFisher Scientific). Aliquots of 0.1 mg/mL were prepared and stored at −80° C.

Real Time Quaking-Induced Conversion (RT-QuIC) Assay

The RT-QuIC reaction buffer was composed of 0.1 M PIPES (pH 6.9), 0.1 mg/ml recombinant α-syn and 10 μM thioflavin-T (Th-T). Reactions were performed in triplicates in a black 96-well microplate with a clear bottom (Nunc) with 85 μl of the reaction mix loaded into each well together with 15 μl of 0.1 mg/ml TBS-soluble fractions. The plate was then sealed with a sealing tape (ThermoFisher) and incubated at 37° C. for 120 h in a BMG FLUOstar OMEGA plate reader with intermittent cycles of 1 min shaking (500 rpm, double orbital) and 15 min rest throughout the indicated incubation time. Th-T fluorescence measurement, expressed as arbitrary relative fluorescence units (RFU), was taken with a bottom read every 15 min using 450±10 nm (excitation) and 480±10 nm (emission) wavelengths. A positive RT-QuIC signal was defined as RFU more than 5 standard deviation units (RFU>5 SD) above the mean of initial fluorescence at 120 h. The sample was considered positive if two or more of the replicates were positive, otherwise the sample was classified as negative.

RT-QuIC Data Analysis

The relative seeding activities of the assayed samples were presented by graphing fluorescence readouts against assay time. For each sample, we calculated three quantitative measures that can be used to analyze the RT-QuIC data: (1) the lag-phase (RFU>5 SD); (2) the amyloid formation rate, expressed as the inverse (1/time to threshold) of the lag-phase (Kang et al., Biomed Res Int. 2017(5413936), 2017), (3) and the maximum fluorescence value (F_(MAX)) measured at the end of the RT-QuIC reaction. For the RT-QuIC negative samples, the lag-phase was assigned as 150 hours.

Tissue Culture of WT BE(2)-M17 Human Neuroblastoma Cells

Human neuroblastoma cells (WT BE(2)-M17) were cultured in Dulbecco's MEM/Nutrient Mix F-12 (1:1) (Hyclone) containing 10% FBS (Hyclone) and 1% penicillin-streptomycin (P/S; 10,000 U/ml penicillin, 10 mg/ml streptomycin, Sigma). The cells were maintained at 37° C. in a humidified incubator with 5% CO₂/95% air.

Tissue Culture of WT SH-SY5Y Human Dopaminergic Neuroblastoma Cells

Human dopaminergic neuroblastoma cells (WT SH-SY5Y) were cultured in Dulbecco's MEM/Nutrient Mix F-12 (1:1) containing 15% FBS, 1% penicillin-streptomycin (P/S; 10,000 U/ml penicillin, 10 mg/ml streptomycin), and supplemented with 1% non-essential MEM amino acid supplement (Gibco) and 2 mM freshly prepared glutamine. The cells were maintained at 37° C. in a humidified incubator with 5% CO₂/95% air.

Measurement of Cell Viability

To assess the cytotoxic effect of different α-syn species, cells were plated at a density of 15,000 cells (100 μl/well) in a 96-well plate. After 24 hours, the medium was replaced with 100 μl of MEM-RS (Hyclone) serum-free medium containing different solutions of α-syn species and treated for 48 hours. A total of 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT) (Sigma-Aldrich) (6 mg/ml) was dispensed into each well, and incubated for 4.5 h. This was replaced with 100 μl/well of lysis buffer (15% SDS, 50% N, N-dimethylformamide, pH 4.7) overnight. The absorbance values at 590 nm were measured in a microplate reader (Perkin Elmer). For experiments assessing the toxic effect of seeded polymerization, cells were treated in serum-free medium containing different solutions of α-syn species (pure fibrils and pure seeds) for 1 hour and monomeric α-syn was then added, followed by 48 hours incubation. Seeds (short fibrils) used in this experiment, were obtained upon sonication of α-syn fibrils.

HEK293-α-syn-EGFP Cell Line Generation

Human embryonic kidney cells 293 (HEK293) were transfected with a plasmid encoding human WT-α-syn fused to EGFP, at the C-terminus, driven by the cytomegalovirus (CMV) promoter. The plasmid contained a selection marker for the antibiotic geneticin (G418) which was used for the selection of the stable transformants. Protein expression was confirmed by western blot analysis and fluorescence microscopy. A clonal HEK293-α-syn-EGFP cell line was selected and used for subsequent experiments. Cells were maintained in DMEM media supplemented with 10% Fetal Bovine Serum Gold (FBS) (PAA) and 1% Penicillin-Streptomycin (PAN). The cells were grown at 37° C. in an atmosphere of 5% CO2. For the seeding experiments, cells were plated on 13 mm glass coverslips in 24-well plates and incubated in 5% FBS-media. The following day, α-syn seeds were prepared in reactions of 150 μl per tube diluted in PBS, fragmented by sonication (Volpicelli-Daley et al., Nat Protoc. 9(9):2135-2146, 2014) and then added to cells at final concentration of 100 nM. Control cells were exposed to vehicle only (PBS). Cells were further incubated for 4 days, washed with PBS and fixed with 4% PFA for 20 min at room temperature (RT), followed by nuclei staining with 4′6′-diamidino-2-phenylindol (DAPI, Sigma-Aldrich, D8417) (1:5000 in DPBS) for 10 min. After a final wash, coverslips were mounted using Mowiol (Sigma-Aldrich) and subjected to fluorescence microscopy. The proportion of cells with α-syn inclusions within the population was then determined by counting. For quantification of aggregation, at least 200 cells were counted per variant and per experiment. Images were acquired using a 63× objective, and analyzed using LAS AF v.2.2.1 (Leica Microsystems) software.

Generation of 4-oxo-2-nonenal (ONE)-, 4-hydroxy-2-nonenal (HNE)-α-syn Oligomers

HNE-/ONE-α-syn oligomers were prepared as previously described (Duffy et al., Free Radic Biol Med. 50(3):428-437, 2011). For generation of HNE-/ONE-α-syn oligomers, α-syn was dialysed against 50 mM disodium hydrogen phosphate, pH 8.5 followed by filtration using 100-kDa MWCO micron spin filter (Millipore) to get rid of high molecular weight aggregates. HNE or ONE (Abcam) was then added to α-syn monomers (140 μM) to get a final molar ratio of 30:1 (HNE/ONE: α-syn) followed by incubation of the samples at 37° C. for 18 hours without shaking. The samples were then centrifuged at 16,900 xg for 5 min to remove any high molecular aggregated species. The supernatant containing the oligomeric species was then purified by size exclusion chromatography on a Superdex 200 gel filtration column (GE healthcare) equilibrated with 20 mM Tris pH 7.4, 0.15 M NaCl buffer. The eluted peaks fractions corresponding to the oligomeric fraction were pooled and quantified using BCA protein assay kit after solubilizing the oligomers in equal volume on 6 M GnHCl.

Filter Retardation Assay

Filter retardation assay was performed using a Minifold 48 slots (GE Healthcare Life Sciences). Each protein (50 μl) at a final concentration of 1 μg/ml was loaded into each slot on a nitrocellulose membrane that has been pre-soaked in PBS. Samples were allowed to absorb onto the nitrocellulose membrane and then slots were washed with 1 ml of PBS. Membranes were then probed with relevant antibodies, and developed with SuperSignal West Pico Chemiluminescent Substrate Kit.

Inhibition ELISA

A 384-well black MaxiSorb microplate (Nunc) was coated with 1 ug/ml of α-syn monomers in 0.2 M NaHCO₃ pH 9.6 with overnight incubation at 4° C. 4B1 antibody at 50 ng/ml was pre-incubated with serial dilutions of α-syn monomers or aggregates with continuous rolling for 2 hours. The antibody-protein mixture was then loaded to the antigen-coated plate and incubated for 10 min at RT. After washing step, goat anti-mouse IgG-RP (1:20,000, Jackson ImmunoResearch) was added for 1 hour to be later detected using SuperSignal ELISA Femto Chemiluminescent Substrate Kit.

Sandwich ELISA

A 384-well ELISA microplate was coated with 4B1 antibody at 0.5 μg/ml overnight at 4° C. in 0.2 M NaHCO₃ pH 9.6. After incubating the plate with 100 μl/well of blocking buffer for 2 hours at 37° C., serial dilutions of α-syn monomers, pS129-α-syn monomers, or different α-syn oligomers were added to corresponding wells, and incubated overnight at 4° C. Biotinylated 11D12 (mouse mAb for total α-syn) (Majbour et al., Mol Neurodegener. 11(7), 2016) was added as detection antibody and incubated at 37° C. for 2 hours followed by a washing step and incubation for 1 hour at 37° C. with streptavidin-HRP (Sigma). The plate was then washed and 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology) was added to corresponding wells. The chemiluminescence, expressed in relative light units, was immediately measured using Envision plate reader (Perkin Elmer Envision).

Tissue Culture of HEK 293 Human Embryonic Kidney Cells

WT HEK293 cells were grown in Dulbecco's MEM-high glucose (Gibco) supplemented by 15% FBS (Gibco) and 1% penicillin-streptomycin (Gibco) and incubated at 37° C. in a 5% CO₂/95% air humidified incubator. After plating HEK cells overnight in 6-well plates, cells were transfected with 2 μg of WT-α-syn plasmid DNA by lipofectamine 3000 reagent (Invitrogen) (Stoppini et al., J Neurosci Methods. 37(2):173-182, 1991). One group of α-syn expressing HEK cells was similarly transfected again with 4 μg of mutant serine 129 to alanine (S129A) seeds the following day.

HEK cells were lysed, at 6, 12, 24, and 48 hours post seed transfection, initially with 1% Trition X-100 in 50 mM Tris, 150 mM NaCl (pH 7.6) containing protease and phosphatase inhibitors to obtain soluble fractions. The pellet was further lysed with 1% SDS in 50 mM Tris, 150 mM NaCl (pH 7.6) with complete inhibitors to attain insoluble fractions. Protein concentration was determined by BCA protein assay (Pierce) prior to analysis on 12% SDS-PAGE and immunoprobing with appropriate antibodies. These include monoclonal antibodies against rabbit pS129-α-syn (AB51253,Abcam), mouse α-syn Syn1 (610786) (BD Biosciences), and non-pS129-α-syn (4B1) in addition to antibody C4 against β-Actin (Sc-47778) (Santa Cruz Biotechnology) to normalize or the amount of proteins. Blots were subsequently incubated with horseradish peroxidase conjugated with anti-rabbit and anti-mouse IgG (Jackson ImmunoResearch), and proteins were detected with LiCOR system.

Organotypic Hippocampal Culture Slices (OHCS)

Organotypic hippocampal slice cultures were created from P7 C57BI6/J mouse pups and injected with S129A-mutated human α-syn pre-formed fibrils as previously described (Elfarash et al., Acta Neuropathol Commun. 7(213), 2019), and cultured according to Stoppini et al. (J Neurosci Methods. 37(2):173-182, 1991). At various time points, tissue was fixed according to Gogolla et al. (Nat Protoc. 1(5):2452-2456, 2006) and stored at 4° C. until all tissue had been collected. Slices were permeabilized in 0.5% triton X-100 in PBS and blocked in 10% BSA. Primary antibodies against pS129-α-syn (D1R1R, Cell Signaling, #23706, 1:1000) and non-pS129-α-syn (4B1, 200 ng/mL) were diluted in 5% BSA and applied overnight at 4° C. After washing 6x 15 min in 1x TBS+0.3% triton X-100, appropriate secondary Alexa-Fluor antibodies were diluted 1:2000, and DAPI 1:1000, in 5% BSA and applied for 3 hours at RT. Washing was repeated and slices were mounted using DAKO Fluorescent Mounting Medium (DAKO, S3023).

Immunofluorescence was evaluated on a Zeiss AxioObserver 7 inverted microscope fitted with an ApoTome to increase Z-plane resolution. For quantification of the distribution of pS129-α-syn vs. WT-α-syn in aggregates, X63 images covering the aggregates were taken (5-20 images/slices depending on the amount of aggregation). Images were analyzed in ImageJ (NIH) in order to compute total phosphorylated and non-phosphorylated aggregate area proportions and mean fluorescence intensity of pS129 aggregates and WT-α-syn aggregates, as well as analyses stratified by aggregate morphology (small, intermediate and long Lewy-neurite-like plus cell body aggregates) and subregion localization.

Wild-Type Mice Injected With Recombinant α-syn Preformed Fibrils (PFFs)

Wild-type C57BI6 mice 2-4 months old (Jackson Laboratory) were housed in the animal facility of the Biomedical Research Foundation at the Academy of Athens in a room with a controlled light-dark cycle (12 hour light-12 hour dark) and free access to food and water. Adult male wild-type C57BI6 mice were subjected to unilateral striatal injections under general isoflurane anesthesia by an apparatus adjusted to the stereotaxic frame (Kopf Instruments). Right dorsal striatum was targeted using the following coordinates from bregma: anteriorposterior+0.5 mm, mediolateral−1.4 mm and dorsoventral in two depths−3.2 mm and −3.4 mm according to mouse brain atlas. A total of 2.6 μg (2 μl ) of mouse recombinant α-syn PFFs were injected at a constant flow rate of 0.3 μl/min. Equal volume of dPBS1x was used for control animals. An interval of 5 min was maintained between the two dorsoventral depths and the needle was slowly removed 5 min after the injection procedure was completed. For immunohistochemical analysis, mice were transcardially perfused under isoflurane anaesthesia, followed by ice-cold 4% paraformaldehyde (PFA), 2 weeks post stereotaxic injections. Following fixation, the brains were dehydrated by sequential incubation in 15% and 30% sucrose, snap frozen in isopentane at −50° C. and stored at −80° C. Free-floating cryostat-cut coronal sections (30 μm) covering the whole nigrostriatal axis were stained with antibodies against pS129-α-syn, 4B1, and DAT. The sections were treated with antigen retrieval solution (citrate buffer, pH=6) at 80° C. for 20 min. To validate whether pS129-positive α-syn accumulations were proteinase K (PK) resistant, sections were incubated with PK (Sigma-Aldrich) 2.5 μg/ml in PBS for 10 min at 25° C. Fluorescent images were obtained in a Leica SP5-II confocal microscope. A protocol with sequential image acquisition was used.

Immunohistochemistry and Immunofluorescence

Formalin-fixed paraffin-embedded brain tissue from the amygdala was obtained post-mortem from patients with PD dementia and DLB. Sections (6 μm) were cut and stained with 4B1, pS129 or pan-α-syn (KM51, Leica Novocastra) antibodies and detected using conventional immunohistochemistry. Regions of interest were imaged on a Zeiss A.1 microscope from five (amygdala) regions and percentage area immunoreactive was evaluated using ImagePro software. The same regions were evaluated on serial sections with different antibodies and compared between cases and across sections, and compared to clinical data obtained during life. 4B1 and pS129 antibodies were co-stained on sections for immunofluorescent analysis using a Leica SP8 confocal microscope. LasX software was used to generate three-dimensional images from z-stacked data to determine whether or not the core of aggregates was phosphorylated at serine 129.

Statistical Analyses

Statistical analyses were done using the Student's t-test for independent variables. Using the GraphPad Prism software (version 8.3.0), statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison test for the MTT cell viability, Th-S, and RT-QuIC assays or followed by the Holm-S̆idák test for the OHCS. The data are presented as mean±standard deviation and represents results from at least 3 independent experiments.

In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. 

1. A hybridoma cell line as deposited with the American Type Culture Collection under patent deposit accession number PTA-127017.
 2. A monoclonal antibody produced by the hybridoma of claim
 1. 3. A hybridoma generated by a method comprising immunizing a mouse with a peptide comprising amino acid residues 125-133 of α-synuclein (SEQ ID NO: 1).
 4. The hybridoma of claim 3, wherein the peptide comprising amino acid residues 125-133 of α-synuclein is CYEMPSEEGY (SEQ ID NO: 3).
 5. A monoclonal antibody produced by the hybridoma of claim
 2. 6. A monoclonal antibody made by a process comprising immunizing a laboratory animal with means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein.
 7. The monoclonal antibody of claim 6, wherein the laboratory animal is a mouse.
 8. The monoclonal antibody of claim 6, wherein the means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein are conjugated to a carrier protein.
 9. The monoclonal antibody of claim 8, wherein the carrier protein is keyhole limpet hemocyanin.
 10. The monoclonal antibody of claim 6, wherein the means for inducing antibodies recognizing aggregated, non-phosphorylated α-synuclein is the peptide CYEMPSEEGY (SEQ ID NO:3).
 11. The monoclonal antibody of claim 2, that is a fragment of a whole antibody.
 12. An immunoassay for the detection or quantitation of aggregated, non-phosphorylated α-synuclein comprising a step for contacting a sample with means for binding WT-α-syn.
 13. The immunoassay of claim 12, wherein the means for binding WT-α-syn is the monoclonal antibody produced by the hybridoma deposited with the American Type Culture Collection under patent deposit accession number PTA-127017. 